Top-Down Proteomics and the Challenges of True Proteoform Characterization.

Top-down proteomics (TDP) aims to identify and profile intact protein forms (proteoforms) extracted from biological samples. True proteoform characterization requires that both the base protein sequence be defined and any mass shifts identified, ideally localizing their positions within the protein sequence. Being able to fully elucidate proteoform profiles lends insight into characterizing proteoform-unique roles, and is a crucial aspect of defining protein structure-function relationships and the specific roles of different (combinations of) protein modifications. However, defining and pinpointing protein post-translational modifications (PTMs) on intact proteins remains a challenge. Characterization of (heavily) modified proteins (>∼30 kDa) remains problematic, especially when they exist in a population of similarly modified, or kindred, proteoforms. This issue is compounded as the number of modifications increases, and thus the number of theoretical combinations. Here, we present our perspective on the challenges of analyzing kindred proteoform populations, focusing on annotation of protein modifications on an "average" protein. Furthermore, we discuss the technical requirements to obtain high quality fragmentation spectral data to robustly define site-specific PTMs, and the fact that this is tempered by the time requirements necessary to separate proteoforms in advance of mass spectrometry analysis.

Considerations for defining +80 Da mass shifts in mass spectrometry-based proteomics: phosphorylation and beyond.

Post-translational modifications (PTMs) are ubiquitous and key to regulating protein function. Understanding the dynamics of individual PTMs and their biological roles requires robust characterisation. Mass spectrometry (MS) is the method of choice for the identification and quantification of protein modifications. This article focusses on the MS-based analysis of those covalent modifications that induce a mass shift of +80 Da, notably phosphorylation and sulfation, given the challenges associated with their discrimination and pinpointing the sites of modification on a polypeptide chain. Phosphorylation in particular is highly abundant, dynamic and can occur on numerous residues to invoke specific functions, hence robust characterisation is crucial to understanding biological relevance. Showcasing our work in the context of other developments in the field, we highlight approaches for enrichment and site localisation of phosphorylated (canonical and non-canonical) and sulfated peptides, as well as modification analysis in the context of intact proteins (top down proteomics) to explore combinatorial roles. Finally, we discuss the application of native ion-mobility MS to explore the effect of these PTMs on protein structure and ligand binding.